EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. The Epidermal Growth Factor Receptor (EGFR), a 170-kD glycoprotein, is composed of an N-terminus extracellular domain, a hydrophobic transmembrane domain, and a C-terminus intracellular region containing the kinase domain. EGFR ligand-induced dimerization activates the intrinsic RTK domain (an Src homology domain 1, SH1), resulting in autophosphorylation on six specific EGFR tyrosine residues in the noncatalytic tail of the cytoplasmic domain.
The cellular effects of EGFR activation in a cancer cell include increased proliferation, promotion of cell motility, adhesion, invasion, angiogenesis, and enhanced cell survival by inhibition of apoptosis. Activated EGFR induces tumor cell proliferation through stimulation of the mitogen-activated protein kinase (MAPK) cascade. Upon ligand binding to the EGFR, the SOS guanine nucleotide exchange factor is recruited to the plasma membrane via the Grb2 adaptor protein, which stimulates the exchange of GTP for GDP on the small G-protein Ras, subsequently activating the MAPK cascade consisting of Raf, MEK, and ERK. Activated ERKs (pMAPK, pERK1/2) in turn phosphorylate and activate transcription factors such as ELK-1 or c-Myc, promoting cell growth.
Multiple growth factor pathways contribute to the progression and survival of NSCLC cells through activation of multiple kinases. The EGFR enhances cancer cell survival also by signaling through the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and the STAT pathway. AKT is stimulated also by other growth factors, including insulin growth factor-1, basic fibroblast growth factor, and interleukins 3 and 6. The three isoforms of AKT 1-3 are all phosphorylated (pAKT) in a similar fashion at residues T308 in the activation domain and S473 in the COOH-terminal domain.
Erlotinib (Tarceva® Genentech/OSI) is a potent epidermal growth factor receptor (HER1/EGFR) tyrosine-kinase inhibitor (TKI) that provides survival benefit to patients with non-small-cell lung cancer (NSCLC) who have failed previous chemotherapy when used as a single agent (WO 01/34574). The efficacy of Tarceva® was studied in various trials. Its chemical name is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine.
The TALENT trial was a placebo-controlled phase III study in first-line NSCLC patients who received gemcitabine and cisplatin (this concurrent chemoradiotherapy was the non-US standard of care) in combination with erlotinib (Tarceva® at 150 mg/day or placebo with. The primary endpoint was survival duration, with secondary endpoints of time to progression, response rate; duration of response; pharmacokinetic and pharmacodynamic parameters, and quality of life. HER1/EGFR and HER2 expression rates were also assessed. A standard safety analysis was done. The overall outcome of the TALENT trial was negative. For the primary and secondary endpoints there was no demonstrable benefit for erlotinib (Tarceva® plus chemotherapy (gemcitabine and cisplatin) compared with gemcitabine and cisplatin alone (Gatzemeier, U., et al., Proc Am Soc Clin Oncol 23 (2004) 617 (Abstract 7010)). Identical results were seen in the US-based TRIBUTE study, with erlotinib plus carboplatin and paclitaxel (Herbst, R. S., et al., J Clin Oncol (2004) ASCO Annual Meeting Proceedings. Post-Meeting Edition; 22 (July 15 Suppl.) (Abstract 7011)). A randomised, placebo-controlled phase III study of single-agent erlotinib as second- or third-line therapy for non-small-cell lung cancer (NSCLC) (BR.21; NCIC/OSIP) found a statistically significant improvement in survival with erlotinib (6.7 months) compared with placebo (4.7 months).
Various studies are related to the investigation of biomarkers in non-small cell lung cancer and their relation to certain EGFR inhibitor drugs. Han, et al., Int J Cancer 113 (2005) 109-115 investigate 65 patients with Gefitinib (Iressa™, EGFR TKI) monotherapy. They analyse EGFR downstream molecules as response predictive markers for gefitinib in chemotherapy-resistant non-small cell lung cancer. Cappuzzo, F. et al., JNCI 96 (2004) 1133-1141 investigate 106 patients with Gefitinib (Iressa; EGFR TKI) monotherapy. They investigate AKT phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer and find that patients with P-AKT-positive tumors who received gefitinib benefited more from the therapy that patients with P-AKT-negative tumors. Vicent, S. et al., Br J Cancer 90 (2004) 1047-1052 investigate 111 NSCLC patients. They find that pERK is activated in non-small-cell lung cancer and associated with advanced tumors. Han, S. W. et al., J Clin Oncol 23 (2005) 2493-2501 investigate 90 patients with Gefitinib (EGFR TKI) monotherapy. They analyse the predictive and prognostic impact of Epidermal Growth Factor Receptor Mutation in Non-Small-Cell lung cancer patients treated with gefitinib. Mukohara, T. et al., Lung Cancer 41 (2003) 123-130 investigate 60 patients, 20 patients per stage who either underwent neoadjuvant chemotherapy or radiation. The EGFR expression correlates with pERK and pAKT expression. The sample size is too low as mentioned by the authors themselves. Raben, D. et al., Int J Radiation Oncology Biol. Phys 59 (2004) 27-38 investigate targeted therapies for non-small-cell lung cancer. Ono, M. et al., Mol Cancer Ther 3 (2004) 465-472 assay 9 NSCLC cell lines and treated with gefitinib. Hirsch, F. R. et al., Curr Opin Oncol 17 (2005) 118-122 review the phosphorylation status of AKT and MAPK as potential marker for gefitinib resistance. Meert, et al., Clinical Cancer Research 9 (2003) 2316-2326 investigate NSCLC cell lines in aspect of EGFR inhibitor activity. Neither EGFR nor Her2 expression levels correlate with sensitivity to EGFR inhibitors. Brognard, J. et al., Cell Death and Differentiation 9 (2002) 893-904 analysed 19 NSCLC cell lines were analysed whereby 17 exhibited phosphorylation of Erkl/2 and constitutive activity. David, 0. et al., Clinical Cancer Research 10 (2004) 6865-6871 disclose that overexpression of pAKT is an independent prognostic factor in NSCLC. Kakiuchi, S. et al., Human Molecular Genetics 13 (2004) 3029-3043 investigate a genome wide cDNA microarray of 33 NSCLC patients. All were given gefitinib in a monotherapeutical setting. No evidence was found for correlation between AKT/pAKT expression level, EGFR gene status or pEGFR staining and gefitinib response. Kim, R. H. et al., Cancer Cell 7 (2005) 263-273 disclosed that DJ-1 expression, an oncogene, equals pAKT level. Balsara, B. R. et al., Carcinogenesis 25 (2004) 2053-2059 investigate 110 NSCLC patients with TMA pAKT expression. No significant difference in survival exists between pAKT negativity and positivity. Hirami, Y. et al., Cancer Letters 214 (2004) 157-164 investigate the relation of epidermal growth factor receptor, pAKT and hypoxia-inducible factor-1 alpha in non-small cell lung cancers. Lee, S. H. et al., APMIS 110 (2002) 587-592 analyses 43 LN metastasis of NSCLC patients. AKT activation in NSCLC plays a role in tumor development rather than progression. Engelman, J. A. et al., Proc. Natl. Acad. Sci. USA 102 (2004) 3788-3793 analyse erbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. David, O., J Cell Mol Med 5 (2001) 430-433 discussess the role of AKT and PTEN as new diagnostics markers in lung cancer. Mantha, A. et al., Clin. Cancer Res. 11 (2005) 2398-2407 investigate the targeting of the mevalonate pathway which inhibits the function of the epidermal growth factor receptor.
Prognostic markers associated with EGFR positive cancer are investigated in WO 2004/046386. Gene expression markers for response to EGFR inhibitor drugs are disclosed by US 2004/0157255. Biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators are disclosed in WO 2004/063709. WO 01/00245 describes humanized anti-ErbB2 antibodies and methods for treating cancer with anti-ErbB2 antibodies, such as humanized anti-erbB2 antibodies.
However, and as suggested by the above cited art, there is still a need to provide methods for determining the sensitivity to EGFR inhibitor therapy, in particular combination therapies of an EGFR inhibitor with a chemotherapeutic agent.